Product name

Anti-PBR antibody [EPR5384]
See all PBR primary antibodies

Description

Rabbit monoclonal [EPR5384] to PBR

Host species

Rabbit

Specificity

IHC results on rat tissues (such as liver and kidney) showed weak cytoplasmic and nuclear staining. However, other customer feedback suggests that this antibody works well in rat. Due to the inconclusive nature of these results, we do not currently guarantee this antibody in rat. Please contact our Scientific support team for more information.

Tested applications

Suitable for: WB,IP,IHC-P,Flow Cyt,ICC/IF

Species reactivity

Reacts with: Mouse, Human

Immunogen

Synthetic peptide within Human PBR aa 150 to the C-terminus (C terminal). The exact sequence is proprietary.
(Peptide available asab170987)

Positive control

• WB: U-87MG, 293T, A431, RAW264.7, and NIH3T3 cell lysates IHC-P: Human bladder carcinoma and colon tissue ICC/IF: U-87MG cells, HeLa cells, TSPO-HAP1 cells Flow Cyt: HepG2 and U-87MG cells. IP: A431 and U-87MG cell lysate.

General notes

 

Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

 

Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab^® patents.

We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

This product is a recombinant rabbit monoclonal antibody.

Form

Liquid

Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.

Storage buffer

PBS 49%,Sodium azide 0.01%,Glycerol 50%,BSA 0.05%

Purity

Protein A purified

Clonality

Monoclonal

Clone number

EPR5384

Isotype

IgG

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PBR knockout HAP1 cell lysate (20 µg)
Lane 3: HEK293 cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab109497 observed at 15 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109497 was shown to specifically react with PBR in wild type HAP1 cells. No band was observed when PBR knockout samples were used. Wild-type and PBR knockout samples were subjected to SDS-PAGE.  Ab109497 and ab8245 (loading control to GAPDH) were diluted at 1/10000 and 1/10000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

TSPO (PBR) expression was abolished in global KO mice without pathological changes

TSPO expression in different tissues from WT and KO mice was detected by western blotting (A) and IHC (B). Scale Bars, 100μm.

4% PFA-fixed tissue sections were blocked with 5% goat serum and incubated overnight at 4°C with ab109497 at 1/1500 dilution. DAB staining.

Note: TSPO and PBR are alternative names for the same target.

(Adapted from Figure 3 of Wang et al)

TSPO (PBR) expression is localized to the mitochondria

(A) Panel shows confocal images of TSPO (red), VDAC1 (green) and nuclear counterstain (blue) in MA-10 Leydig cells. (B) Negative control panel. Colocalization of TSPO to the mitochondrial protein VDAC1 validates the specific localization of TSPO. Scale bar 20 µm.

Cells were fixed with 4% formaldehyde and permeabilized using 0.1% Triton X-100. Cells were then blocked using 5% normal goat serum and incubated with ab109497 at 1/200 dilution.

Note: TSPO and PBR are alternative names for the same target.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human bladder carcinoma tissue labelling PBR with purified ab109497 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

ab109497 staining PBR in HeLa. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109497 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Flow Cytometry analysis of U87-MG cells labelling PBR with purified ab109497 at 1/150 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab109497 (purified) at 1/60 immunoprecipitating PBR in A431 whole cell lysate.

Lane 1 (input): A431 whole cell lysate (10µg)

Lane 2 (+): ab109497 + A431 whole cell lysate (10µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109497 in A431 whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

ab109497 staining PBR in wild-type HAP1 cells (top panel) and PBR knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109497 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunocytochemistry/Immunofluorescence analysis of U87-MG cells labelling PBR with purified ab109497 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

All lanes : Anti-PBR antibody [EPR5384] (ab109497) at 1/50000 dilution (purified)

Lane 1 : RAW264.7 cell lysate
Lane 2 : NIH/3T3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 19 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PBR antibody [EPR5384] (ab109497) at 1/50000 dilution (purified)

Lane 1 : U87-MG cell lysate
Lane 2 : HEK293 cell lysate
Lane 3 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 19 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

ab109497 (purified) at 1/60 immunoprecipitating PBR in U87-MG whole cell lysate.

Lane 1 (input): U87-MG whole cell lysate (10µg)

Lane 2 (+): ab109497 + U87-MG whole cell lysate (10µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109497 in U87-MG whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling PBR with unpurified ab109497 at a dilution of 1/100.

Overlay histogram showing HepG2 cells stained with unpurified ab109497 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified ab109497, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.